For Research Use Only
Corning Life Sciences – Discovery Labware
Patent Pending
Corning® TransportoCells™
Cryopreserved Transporter Cells
Instructions for Use
SLC transporters are multi-transmembrane-bound proteins abundantly expressed in tissues important for drug
disposition and elimination, such as liver and kidney, where they play a key role in facilitating the uptake of a variety of
solutes including nutrients, environmental toxins, drugs, and other xenobiotics. It is now well recognized that alteration
of certain SLC transporter functions by factors such as genetic polymorphism or drug-drug interactions can lead to
altered pharmacokinetics and, potentially, toxicity. The FDA and EMA drug-drug interactions (DDI) guidance
documents recommend evaluating specific SLC transporters for their potential for DDI.
Corning® TransportoCells™ cryopreserved transporter cells are mammalian cells (HEK-293) transiently overexpressing a single human SLC transporter protein. The product is a convenient, easy to use consumable for
measuring drug uptake by SLC transporters. Kinetic analyses of drug uptake, as well as inhibition studies for DDI, are
easily carried out using a variety of detection systems including scintillation counting for radiolabelled compounds,
mass spectrometry for cold compounds, and fluorescence detection for fluorogenic compounds. The
TransportoCells™ product portfolio includes the following SLC transporters: Organic Anion-transporting Polypeptide
1B1, 1B3 2B1, and 1A2 (OATP1B1, OATP1B3, OATP2B1, and OATP1A2), Organic Anion Transporters 1 and 3
(OAT1 and OAT3), Organic Cation Transporters (OCT1 and OCT2), Multidrug and Toxin Extrusion Transporters
(MATE1 and MATE2-K), Peptide Transporter 1 and 2 (PEPT1 and PEPT2) and Na+-Taurocholate Co-transporting
polypeptide (NTCP)
This Instruction for Use describes the procedure for performing an uptake assay with the Corning TransportoCells™
products. One vial contains 10 million viable cells for one 24-well plate, one 48-well plate, or one 96-well microplate
when the procedure is followed.
Products
Cat. No.
Transporter
Full Name
354859
OATP1B1*1a/SLCO1B1*1a
354851
354857
354858
354852
354853
354855
354856
354860
354861
354862
354863
354864
354854
OATP1B3/SLCO1B3
OAT1/SLC22A6
OAT3/SLC22A8
OCT1/SLC22A1
OCT2/SLC22A2
MATE1/SLC47A1
MATE2-K/SLC47A2
PEPT1/SLC15A1
PEPT2/SLC15A2
OATP2B1/SLCO2B1
OATP1A2/SLCO1A2
NTCP/SLC10A1
Control
Organic Anion-transporting Polypeptide 1B1
Wild Type
Organic Anion-transporting Polypeptide 1B3
Organic Anion Transporter 1
Organic Anion Transporter 3
Organic Cation Transporter 1
Organic Cation Transporter 2
Multidrug and Toxin Extrusion Transporter 1
Multidrug and Toxin Extrusion Transporter 2-K
Peptide Transporter 1
Peptide Transporter 2
Organic Anion-transporting Polypeptide 2B1
Organic Anion-transporting Polypeptide 1A2
Na+-Taurocholate Co-transporting polypeptide
Empty Expression Vector
Gene Accession
Number
NM_006446
NM_019844
NM_004790
NM_004254
NM_003057
NM_003058
NM_018242
NM_001099646
NM_005073
NM_021082
NM_007256
NM_021094
NM_003049
N/A
Discovery Labware, Inc., Two Oak Park, Bedford, MA 01730, USA. Tel: +1.978.442.2200 (U.S.)
[email protected] www.corning.com/lifesciences
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
For a listing of trademarks, visit www.corning.com/lifesciences/trademarks
© 2015 Corning Incorporated
For Research Use Only
Corning Life Sciences – Discovery Labware
Patent Pending
Reagents and Disposables
Description
Supplier
Catalog No.
Dulbecco's Modified Eagle’s Medium (DMEM), high glucose
MEM Non-essential Amino Acid Solution (100 X)
Fetal Bovine Serum (FBS) (NOTE: Heat inactivation is not
recommended)
Corning Life Sciences
Corning Life Sciences
10-017-CV
25-025-CI
Corning Life Sciences
35-010-CV
Hank’s Balanced Salt Solution (HBSS) with Ca2+ and Mg2+ (1 X)
Corning Life Sciences
21-023-CV
Corning BioCoat™ 24-well Poly-D-Lysine plate
Corning BioCoat™ 48-well Poly-D-Lysine plate
Corning Life Sciences
Corning Life Sciences
354414
354509
Corning BioCoat™ 96-well Poly-D-Lysine plate
Corning Life Sciences
354461
Corning 96-well Poly-D-Lysine plate, black/clear
Falcon® 96-well assay plate, clear
Corning Life Sciences
Corning Life Sciences
356640
353075
500 mM Sodium Butyrate Solution, filter sterile
1M HEPES
EMD Millipore
Life Technologies
TR-1008-G
15630-080
Mammalian Protein Extraction Reagent (M-PER)
Bicinchoninic Acid (BCA) Protein Assay Kit
Thermo-Scientific
Thermo Scientific
78503
23225
MES
Ammonium Chloride
Sigma-Aldrich
Sigma-Aldrich
M3671
A9434
Dimethyl Sulfoxide (DMSO)
Acetonitrile
Sigma-Aldrich
Sigma-Aldrich
D8418
9070-01
Materials and Equipment
Tissue culture hood
Orbital rotator
37ºC CO2 cell culture incubator
Water bath
Vacuum aspirator
Centrifuge
Cell counter
Detection system options:
- Scintillation counter/microbeta scintillation counter
- LC-MS
- Fluorescence reader
Pipet aid and 5, 10, 25 mL pipets
Multichannel pipettor and corresponding tips
Pipets: 10, 20, 200, 1000 µL pipets and corresponding tips
0.2 µm sterilizing bottle filter
Sample preparation conical tubes and vials
Reagent Reservoir
Ice bucket
Discovery Labware, Inc., Two Oak Park, Bedford, MA 01730, USA. Tel: +1.978.442.2200 (U.S.)
[email protected] www.corning.com/lifesciences
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
For a listing of trademarks, visit www.corning.com/lifesciences/trademarks
© 2015 Corning Incorporated
For Research Use Only
Corning Life Sciences – Discovery Labware
Patent Pending
ASSAY PROCEDURE
I.
Prepare reagent
Prepare plating media:
Reagent
Quantity
DMEM (high glucose)
445 mL
MEM non-essential amino acid solution (100X)
5 mL
Fetal bovine serum (FBS)
50 mL
o
Sterilize the media using a 0.2 µm filter, then store at 4 C for up to 2 weeks.
Prepare HBSS buffer with 10 mM HEPES, pH 7.4 (refer to Uptake buffer in the following procedure):
Reagent
Quantity
HBSS buffer
445 mL
1M HEPES
5 mL
o
Adjust pH to 7.4 and store at 4 C for up to 3 months.
Prepare HBSS buffer with 5 mM MES, pH 6.0 (buffer used for PEPT1, PEPT2 and OATP1A2):
Reagent
Quantity
HBSS buffer
500 mL
MES powder
0.5 g
o
Adjust pH to 6.0 and store at 4 C for up to 3 months.
Prepare HBSS containing 40 mM Ammonium Chloride (for MATE1 and MATE2-K):
Reagent
Quantity
HBSS with 10 mM HEPES
100 mL
Ammonium Chloride powder
214 mg
Adjust pH to 7.4 and prepare freshly before use.
Discovery Labware, Inc., Two Oak Park, Bedford, MA 01730, USA. Tel: +1.978.442.2200 (U.S.)
[email protected] www.corning.com/lifesciences
For research use only. Not for use in diagnostic or therapeutic procedures.
For a listing of trademarks, visit www.corning.com/lifesciences/trademarks
® 2014Corning Incorporated
For Research Use Only
Corning Life Sciences – Discovery Labware
Patent Pending
II.
Thaw and plate cells
Must read before starting:
A. A CO2 incubator with low or no humidity (i.e., remove water pan from
incubator) is recommended.
B. Cell plates should be placed at the center of the shelf in the CO2 cell
culture incubator to avoid media evaporation.
C. To achieve consistent cell attachment and avoid cell peeling during the
assay, do not use heat-inactivated serum for plating the cells.
D. Refer to the table below for the recommended cell seeding density and
liquid handling volumes for different plate formats (24-well, 48-well, and 96well).
E. All the reagents used for cell culture must be sterile.
24-well
48-well
96-well
Seeding density for OATPs,
OATs, OCTs, PEPTs, NTCP,
and Control
350K – 400K
175K – 200K
100K
Seeding density for MATEs
400K
200K
100K
Re-feeding media volume
400 µL
200 µL
150 µL
Wash buffer volume
400 µL
200 µL
200 µL
Uptake compound solution
300 – 400 µL
150 – 200 µL
120 µL
Lysis buffer solution
400 µL
200 µL
120 µL
Sample size for uptake analysis
300 µL
125 µL
50 µL
Sample size for protein analysis
25 µL
25 µL
25 µL
The following thawing and plating and assay procedure is for use with 400,000 cells per
well in a 24-well plate and 100,000 cells per well in a 96-well microplate. For other seeding
densities or plate formats, refer to the table above.
1. Warm the plating media in a 37oC water bath.
2. For one vial of cells, aliquot 20 mL of warmed plating media into a 50 mL conical centrifuge tube.
Discovery Labware, Inc., Two Oak Park, Bedford, MA 01730, USA. Tel: +1.978.442.2200 (U.S.)
[email protected] www.corning.com/lifesciences
For research use only. Not for use in diagnostic or therapeutic procedures.
For a listing of trademarks, visit www.corning.com/lifesciences/trademarks
® 2014Corning Incorporated
For Research Use Only
Corning Life Sciences – Discovery Labware
Patent Pending
3. Remove the cells from the liquid nitrogen.
o
4. Thaw the cryovials in a 37 C water bath for 2 minutes by gently moving the vial back and forth in the
water bath to facilitate heat transfer, until there is a small ball of frozen cells remaining in the vial.
5. Transfer the thawed cells into the 20 mL of pre-aliquot plating media. Centrifuge at 100 g for 10
minutes.
6. Aspirate the supernatant carefully without disturbing cell pellet and resuspend cells in 5 mL of plating
media.
7. Take cell samples and mix well with Trypan Blue (or other cell counting dye) by vortexing for 5 to 10
seconds. Then count the cells and determine the viability and viable cell number.
8. Add the plating media to the cell suspension to achieve the final viable cell concentration of 1 x 106
cells per mL.
9. For a 24-well plate, add 400 µL of cell suspension into each well for a seeding density of 400K cells
per well. A sample plate layout is shown in Appendix I. Gently rock the plate in different directions
(i.e., front to back, right to left and across the diagonals) to evenly distribute the cells.
10. For a 96-well microplate, add 100 µL of cell suspension into each well for a seeding density of 100K
cells per well. A sample plate layout is shown in Appendix I. Gently rock the plate in different
directions (i.e., front to back, right to left and across the diagonals) to evenly distribute the cells.
11. Incubate the plate at 37oC with 8% CO2 with low or no humidity.
12. After incubation for 3 to 4 hours, re-feed the cells with fresh plating media or plating media
supplemented with sodium butyrate as suggested below. Aspirate the plating media and replace with
400 µL of pre-warmed media for a 24-well plate or 150 µL for a 96-well microplate.
NOTE: OATP1B1, OATP1B3, OATP1A2, MATEs, PEPTs, and NTCP require sodium butyrate for
optimal uptake activity.
For these TransportoCells products and corresponding control cells, re-feed the cells with plating
media supplemented with 2 mM sodium butyrate at Step 12 (i.e., add 40 µL of 500 mM sodium
butyrate solution into 10 mL of plating media).
OATP2B1, OATs, and OCTs do not need sodium butyrate treatment.
o
13. Incubate the cells overnight at 37 C with 8% CO2 with low or no humidity.
III.
Uptake Assay for 24-well or 96-well microplates (24 hours post-plating)
1. Warm up one bottle of Uptake buffer in a 37oC water bath, and chill the other bottle on ice.
2. Prepare test/control compound as described below at the desired concentration. The recommended
positive control substrate information is listed in Appendix II.
OATP1B1, OATP1B3, OATP2B1,
OAT1, OAT3, OCT1, OCT2,
MATE1, MATE2-K, and NTCP
Prepare test/control compound in Uptake buffer
PEPT1, PEPT2, and OATP1A2
Prepare test/control compound in HBSS buffer
with 5 mM MES, pH 6.0
3. Warm the compound solution in a 37oC water bath/incubator for 10 minutes.
Discovery Labware, Inc., Two Oak Park, Bedford, MA 01730, USA. Tel: +1.978.442.2200 (U.S.)
[email protected] www.corning.com/lifesciences
For research use only. Not for use in diagnostic or therapeutic procedures.
For a listing of trademarks, visit www.corning.com/lifesciences/trademarks
® 2014Corning Incorporated
Corning Life Sciences – Discovery Labware
For Research Use Only
Patent Pending
4. Wash the cells twice with 400 µL (24-well plate) or 200 µL (96-well microplate) of pre-warmed
Uptake buffer per well. Add 400 µL (24-well plate) or 200 µL (96-well microplate) of pre-warmed
Uptake buffer into each well, and incubate the plate at 37oC for 10 minutes.
NOTE: For MATE1 and MATE2-K, proceed to Step 5; for others, skip Step 5 and proceed to Step 6.
5. Aspirate the buffer and add 400 µL (24-well plate) or 200 µL (96-well microplate) of pre-warmed
Uptake buffer containing 40 mM ammonium chloride into each well, and incubate at 37oC for 20
minutes. At the end of the incubation, wash once with pre-warmed Uptake buffer.
6. After the incubation is complete, carefully aspirate the buffer completely. Add 300 µL (24-well plate)
or 120 µL (96-well microplate) of pre-warmed compound solution into each well and incubate at
37oC for the desired amount of time.
7. At the end of incubation, stop the incubation by placing the plates on ice and aspirating the
incubation buffer immediately.
8. Wash the cells two times with 400 µL (24-well plate) or 200 µL (96-well microplate) of pre-chilled
Uptake buffer per well.
9. After the second wash, carefully aspirate the buffer completely.
For radiolabeled or fluorogenic compound, proceed with Steps 10 to 13
10. Add 400 µL (24-well plate) or 120 µL (96-well microplate) of M-Per Mammalian Protein Extraction
Reagent. Cover the plate and gently shake the plate on the orbital shaker at low speed (i.e., 50-100
rpm) for 5 minutes at room temperature. For fluorogenic compound, the plate is ready for analysis
on a fluorescence reader. For radiolabeled compound, continue to proceed with Steps 11 to 13.
11. Take 300 µL (24-well plate) or 50 µL (96-well microplate) of cell lysate and add into a scintillation
vial containing 5 mL of scintillation fluid. Take 300 µL (24-well plate) or 50 µL (96-well microplate) of
compound solution and add into scintillation vials for normalization
12. For a 96-well scintillation counter, add 50 µL of cell lysate into a 96-well scintillation plate containing
250 µL of scintillation fluid.
13. Samples are ready for analysis on the scintillation counter.
For non-radiolabeled compound, proceed with Steps 14 to 19
14. Add 400 µL (24-well plate) or 120 µL (96-well microplate) of 80% acetonitrile containing LC-MS/MS
internal standard into each sample well. Cover the plate and gently shake the plate on the orbital
shaker at low speed (i.e., 50 to 100 rpm) for 20 minutes at room temperature.
15. For a 24-well plate assay, transfer 300 µL of cell lysate into a 96-well microplate; for a 96-well
microplate assay, proceed to Step 16.
16. Centrifuge the 96-well polypropyrene microplate at 4000 rpm for 20 min.
17. Transfer 50 µL of supernatant into a LC-MS injection plate.
18. Add 50 µL of dH2O into each well.
19. Analyze the sample on a LC-MS system.
IV.
Protein Assay
20. After the cells are lysed by M-Per Mammalian Protein Extraction Reagent, take duplicate 25 µL
aliquots from each sample well for protein analysis using a BCA protein assay kit following the
manufacturer’s procedure.
Discovery Labware, Inc., Two Oak Park, Bedford, MA 01730, USA. Tel: +1.978.442.2200 (U.S.)
[email protected] www.corning.com/lifesciences
For research use only. Not for use in diagnostic or therapeutic procedures.
For a listing of trademarks, visit www.corning.com/lifesciences/trademarks
® 2014Corning Incorporated
Corning Life Sciences – Discovery Labware
For Research Use Only
Patent Pending
SAFETY RECOMMENDATIONS:
Safety assessment indicates this product is not hazardous, therefore no SDS (Safety Data Sheet) is provided. It is
recommended that users handle reagents as potentially biohazardous material under Biosafety Level 2 containment.
All reagents should be handled in accordance with good industrial hygiene and laboratory safety practices.
Use of Genetically Modified Microorganisms (GMM)
Information for European Customers: These products are genetically modified microorganisms as described in Corning
Life Sciences technical literature. As a condition of sale, use of this product must be in accordance with all applicable
local guidelines on the contained use of genetically modified microorganisms, including the Directive 2009/41/EC of
the European Parliament and of the Council.
DISCLAIMER
It is the Customers responsibility to use TransportoCells™ in a way that does not infringe a third party’s intellectual
property rights. Corning is not liable for any use of TransportoCells™ that infringes third party intellectual property
rights (deliberate or otherwise). By using this product you agree to indemnify Corning against all damages or losses
suffered or incurred as a result of your use of TransportoCells™.
Discovery Labware, Inc., Two Oak Park, Bedford, MA 01730, USA. Tel: +1.978.442.2200 (U.S.)
[email protected] www.corning.com/lifesciences
For research use only. Not for use in diagnostic or therapeutic procedures.
For a listing of trademarks, visit www.corning.com/lifesciences/trademarks
® 2014Corning Incorporated
Corning Life Sciences – Discovery Labware
For Research Use Only
Patent Pending
Appendix I: PLATE MAP
24-well plate
Km Assay (8 concentrations in triplicate)
IC50 Assay (8 concentrations in triplicate)
96-well microplate
Km Assay (8 concentrations in triplicate)
IC50 Assay (8 concentrations in triplicate)
For Research Use Only
Corning Life Sciences – Discovery Labware
Patent Pending
Appendix II: POSITIVE CONTROL SUBSTRATES
Cat. No.
Transporters
Sodium
Butyrate
Conc.
(mM)
Substrate
Fluorescein
methotrexate (FMTX)
Wavelength:
Ex:485 nm; Em: 528 nm
Cholecystokinin
Octapeptide
(CCK-8)
Ordering info
(cold compound)
Ordering info
(radiolabeled
compound)
% of Hot
Final
Conc.
(µM)
Assay
Incubation
Time (min)
Life Technologies
Cat. No. M1198MP
N/A
N/A
5.0
10 min
Sigma-Aldrich
Cat. No. C2175
PerkinElmer
Cat. No. NET11620
1%
2.0
5 min
10%
3.0
10 min
1%
2.0
5 min
354859
OATP1B1*1a
2
354851
OATP1B3
2
354857
OAT1
N/A
p-Aminohippuric Acid
(PAH)
Sigma-Aldrich
Cat. No. A3759
354858
OAT3
N/A
Estrone-3-Sulfate (E3S)
Sigma-Aldrich
Cat. No. E0251
354852
OCT1
354853
OCT2
354855
MATE1
354856
MATE2-K
354860
PEPT1
2
354861
PEPT2
2
354862
OATP2B1
354863
354864
American Radiolabeled
Chemicals (ARC)
Cat. No. ART 0209
PerkinElmer
Cat. No.
NET203250UC
10 min
N/A
Tetraethylammonium
(TEA)
Sigma-Aldrich
Cat. No. T2265
American Radiolabeled
Chemicals (ARC)
Cat. No. ARC 1540
100%
30
2
2 min
Glycyl-sarcosine
(GlySar)
Sigma-Aldrich
Cat. No. G3127
Moravek
Cat.No. MT-1545
1%
50
5 min
N/A
Estrone-3-Sulfate (E3S)
Sigma-Aldrich
Cat. No. E0251
PerkinElmer
Cat. No. NET203
1%
2
5 min
OATP1A2
2
Estrone-3-Sulfate (E3S)
2
5 min
2
Taurocholic acid (TCA)
PerkinElmer
Cat. No. NET203
PerkinElmer
Cat. No. NET322
1%
NTCP
Sigma-Aldrich
Cat. No. E0251
Sigma-Aldrich
Cat. No. T4009
1%
2
5 min
For Research Use Only
Corning Life Sciences – Discovery Labware
Patent Pending
Appendix III: TROUBLESHOOTING GUIDE
Problem
Low viability after thawing
Cause
Cells are counted incorrectly
Solution
Make sure the cell suspension is homogeneous. Take another sample
and count again.
Cells are not stored correctly
Re-order the cells and store in liquid nitrogen freezer immediately upon
receiving. Keep in liquid nitrogen until thawing.
Cells are thawed improperly
Cells should be thawed quickly in a 37 C water bath. Carefully follow
procedure in the Instruction for thawing cells.
Use plating media listed in the “Instruction for Use” to thaw and plate the
cells. No extra purification step is needed prior to seeding.
Plating medium is not correct
o
NOTE: The products are tested to have equal or greater than 80% post-thaw viability. In the case of improper handling, continue to plate and assay if the viability is 70% and <80%. Discontinue
the experiment if the viability is below 70%. Our data indicate that cells with 70% to 80% viability perform reasonably well in the uptake assay but with compromised 24-hour cell confluency.
Low cell recovery after thawing
Lose cells during aspiration
Cell suspension is not homogeneous
Cell clumps are not dissociated
completely during re-suspension
Carefully aspirate the supernatant and leave 0.5 ml to 1.0 mL
supernatant during aspiration.
Re-suspend the cell pellet thoroughly and re-count.
The clumps with 2 to 5 cells are typically observed during the thawing
process. If clumps with more than 10 cells are observed, re-suspend the
cell suspension five more times. Take a small amount of cells out for cell
counting. Vortex the cells for 10 seconds to ensure all large cell clumps
are dissociated before adding cell counting dye.
NOTE: One vial of Corning® TransportoCells™ product provides a minimum of 10 million viable cells following the thawing procedure in the “Instruction for Use.” Typically 10 to 15 million viable
cells can be obtained per vial. If the recovery per vial is more than 15 million, re-check the cell counting to ensure proper seeding density.
Cells form a sparse and patchy
monolayer with sparse area
Low uptake activity/uptake ratio
Cells are not evenly distributed at
plating step
Low seeding density
Addition of sodium butyrate may impact
the cell attachment slightly
Cells are cultured for more than 48
hours
Assay condition is not optimal
Inconsistent uptake activity/uptake ratio
Cells are washed off during assay
Inconsistent assay materials
Follow the instruction for plating, and gently shake the plates several
times to ensure the cells evenly distributed.
Check the cell counting and plate the cells at the recommended seeding
density in the “Instruction for Use.”
Sodium butyrate can be omitted for improved cell confluence but activity
will be reduced.
Ideally use the cells within 24 to 48 hours after plating. Transporter
protein expression and activity start to diminish quickly 48 hours postplating, even more after subculturing the cells.
Check the condition of substrate. Follow the “Instruction for Use” to
perform uptake assay.
Cell monolayer is too confluent. Check the cell counting to ensure the
correct seeding density or optimize the seeding density.
Check the humidity level of cell culture incubator; remove the water pan
to lower the humidity.
Use the recommended FBS, do not heat inactivate the serum.
Check the plates before assay and ensure there is no significant media
evaporation; place the plates at the center of the shelf, avoid to be too
close to the wall of the incubator.
Carefully wash and aspirate without disrupting the cell monolayer.
Measure protein content per sample well to normalize the result.
Use the assay materials from the same supplier, particularly the
radiolabeled probe substrates.
Corning Life Sciences – Discovery Labware
For Research Use Only
Patent Pending